Quantification of impurities for release testing of peptide products

ABSTRACT

The present invention relates to a method for the quantitative determination of an impurity present in a peptide product, wherein the impurity cannot be separated from other impurities or the main product. The method particularly involves the use of high resolution mass spectrometry (MS) detection with or without high performance liquid chromatography (HPLC). The method can be used for the investigation of the quality of peptides and proteins, particularly of pharmaceutical peptides and proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/870,288, filed Apr. 25, 2013, which claims the benefit ofInternational Patent Application No. PCT/EP2012/057771 filed on Apr. 27,2012 and U.S. Provisional Application No. 61/665,098 filed on Jun. 27,2012, the entire contents of each of which are incorporated herein byreference.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in the ASCII text file, named as28977_SequestListing.txt of 3 KB, created on Apr. 22, 2013, andsubmitted to the United States Patent and Trademark Office via EFS-Web,is incorporated herein by reference.

DESCRIPTION

The present invention relates to a method for the quantitativedetermination of an impurity present in a peptide product, wherein theimpurity cannot be separated from the main product and optionally fromother impurities. The method particularly involves the use of highresolution mass spectrometry (MS) detection with or without highperformance liquid chromatography (HPLC). The method can be used for theinvestigation of the quality of peptides and proteins, particularly ofpharmaceutical peptides and proteins, and formulations thereof.

Using well-known recombinant DNA and chemical solid phase synthesisprocesses, several proteins and peptides have been synthesized forpharmaceutical use. The production of these proteins and peptides,however, often leads to a multiplicity of undesired synthesisby-products. This is especially the case when they are produced by solidphase synthesis. With an increase in length of the peptide/protein,leading to an increase in the synthesis steps, these by-products may bepresent in 50 to 70% of the crude product.

Thus, an important part of the synthesis is the purification of the mainproduct from the synthesis by-products. This purification is mainly doneby chromatographic procedures. For use as an active ingredient in apharmaceutical product, the final purified peptide product needs to beanalyzed with regard to the purity, which is also done mostly usingchromatographically procedures. Since the differences in the molecularstructure of the desired peptide product and the undesired synthesisby-products are often very minor compared with the overall structure,the chromatographic properties of these products may be identical ornearly identical. This often leads to a co-elution of these impuritieswith other impurities and the main product itself in a chromatographicseparation procedure.

The development of selective analytical methods is one of the keyaspects for the release testing of peptide product compositions used aspharmaceutical compounds. But due to the reasons described above, evengreat efforts may not result in the separation of all relevantimpurities by chromatographic methods. Thus, there is a need to providenovel methods for determining the amounts of impurities in peptideproducts, particularly the amount of impurities which cannot bequantitatively separated from the desired main product bychromatographic methods.

The present invention provides a method for the quantitativedetermination of impurities in peptide product compositions, which cannot be separated chromatographically from the main product or whichco-elute with other impurities or ingredients of the composition. Thismethod is shown exemplarily for the peptide Lixisenatide (AVE0010), aGLP-1 agonist having a length of 44 amino acids long. The amino acidsequence of Lixisenatide is shown in SEQ ID NO:1:

H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-I-E-W-L-K-N-G-G-P-S-S-G-A-P-P-S-K-K-K-K-K-K-NH₂

Lixisenatide is produced by a chemical solid phase synthesis process. Bymeans of chromatographic purification procedures, most of the impuritiescan be separated from the desired main product.

But nevertheless two impurities, Di-Ser(33)-AVE0010 andDi-Ala(35)-AVE0010, cannot be separated by chromatographic methods. Theamino acid sequences of these impurities are as follows:

Di-Ser(33)-AVE0010 (SEQ ID NO: 2)H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-I-E-W-L-K-N-G-G-P-S-S-S-G-A-P-P-S-K-K-K-K-K-K-NH₂ Di-Ala(35)-AVE0010(SEQ ID NO: 3) H-G-E-G-T-F-T-S-D-L-S-K-Q-M-E-E-E-A-V-R-L-F-I-E-W-L-K-N-G-G-P-S-S-G-A-A-P-P-S-K-K-K-K-K-K-NH₂

Since Lixisenatide is used as a pharmaceutical product, particularly inthe treatment of diabetes patients, the presence and the amount of theabove impurities has to be determined in order to allow batch release.The present inventors have now found that a quantitative determinationof the above impurities may be achieved using high resolution massspectrometric techniques with or without prior chromatography.

A subject matter of the present invention is a method for thequantitative determination of an impurity present in a peptide productcomposition, comprising the steps:

-   -   (a) providing a peptide product composition comprising a peptide        product and an unknown amount of at least one impurity, wherein        said impurity cannot be separated from the peptide product or        another ingredient of the composition by a chromatographic        procedure,    -   (b) providing at least one sample of the peptide product        composition without said impurity added and optionally at least        one further sample of the composition with a known amount of        said impurity added,    -   (c) quantitatively determining said impurity in said at least        one sample from step (b) by mass spectrometry, and    -   (d) calculating the amount of said impurity in the peptide        product composition based on the results of (c).

A further subject matter of the present invention is a method for thequantitative determination of an impurity present in a peptide productcomposition, comprising the steps:

-   -   (a) providing a peptide product composition comprising a peptide        product and an unknown amount of at least one impurity, wherein        said impurity cannot be separated from the peptide product or        another ingredient of the composition by a chromatographic        procedure,    -   (b) providing at least three samples of the peptide product        composition, wherein a first sample comprises the peptide        product composition without said impurity added, and wherein at        least two further samples comprise the peptide product        composition each with a different known amount of said impurity        added,    -   (c) quantitatively determining said impurity in said at least        three samples from step (b) by mass spectrometry and    -   (d) calculating the amount of said impurity in the peptide        product composition based on the results of (c).

A further subject matter of the invention is a reagent kit fordetermining the amount of impurities in a Lixisenatide (AVE 0010)product composition, comprising:

-   -   (i) at least one stock preparation of Di-Ser(33)-AVE0010 and/or    -   (ii) at least one stock preparation of Di-Ala(35)-AVE0010.

Still, a further subject-matter of the invention is a method for thequality control of a composition comprising an exendin peptide productcomprising the amino acid sequence S-S-G-A, preferably of a compositioncomprising a Lixisenatide (AVE0010) product, comprising quantitativelydetermining the amount of a Di-Ser(33)-peptide, e.g. Di-Ser(33)-AVE0010and/or a Di-Ala(35)-peptide, e.g. Di-Ala(35)-AVE0010, in saidcomposition.

The present invention relates to the determination of an impuritypresent in a peptide product composition. The term “peptide product”encompasses peptides and proteins having a length of at least 5 or atleast 10 amino acids and up to 50 or up to 100 amino acids or evenlonger. The peptide product may consist of genetically encoded aminoacid building blocks or may comprise non-genetically encoded amino acidbuilding blocks, e.g. non-naturally occurring amino acids, D-amino acidsor chemically modified amino acids or may consist of several peptidechains linked e.g. by disulfide bridges. The peptide product may furthercontain modifications at the N- and/or C-terminus and/or at side chains,e.g. an acylation, an amidation or the addition of non-peptide sidechain groups such as lipophilic groups. The peptide product may belinear or circular. Preferably, the peptide product has a length from5-100 amino acids.

The synthesis of the peptide product may involve recombinant DNAprocesses and/or chemical synthesis processes. Preferably, the peptideproduct has been chemically synthesized, particularly by a solid phasesynthesis procedure which is well-known in the art, e.g. a procedureinvolving a stepwise elongation of a peptide chain coupled to a carrier,e.g. a synthetic resin. In a preferred embodiment of the invention, thepeptide product is an Exendin peptide, e.g. Exendin-4, Liraglutide orLixisenatide (AVE0010). Further examples of peptide products areinsulins and insulin analogues or DPP-4 inhibitors. More preferably, thepeptide product is an Exendin peptide, e.g. an Exendin peptidecomprising the amino acid sequence S-S-G-A, most preferably Lixisenatide(AVE0010).

The method of the present invention involves the quantitativedetermination of an impurity present in a peptide product after itssynthesis, e.g. after its synthesis by a solid phase synthesis procedureor as a degradation product after storage. The impurity is usually apeptide impurity, e.g. a peptide which differs from the desired mainpeptide product in at least one amino acid building block and/or achemical modification. For example, the impurity may be a peptide whichdiffers from the desired peptide product by at least one dimeric aminoacid building block, i.e. a Di-serine or Di-alanine building block. Inthe case of an Exendin peptide, e.g. an Exendin peptide comprising theamino acid sequence S-S-G-A, the impurity may be e.g. aDi-Ser(33)-exendin peptide, a Di-Ala(35)-exendin peptide or combinationsthereof. In the case of Lixisenatide, the impurity may e.g. beDi-Ser(33)-AVE0010, Di-Ala(35)-AVE0010 or combinations thereof.

The method of the present invention comprises the quantitativedetermination of at least one impurity in a peptide product composition.The peptide product composition comprises at least one peptide productand at least one impurity to be determined. Further, the composition maycomprise other ingredients, e.g. peptide or non-peptide ingredientsincluding other impurities. The peptide product composition may e.g. bea pharmaceutical formulation or a composition intended for themanufacture of a pharmaceutical formulation, e.g. a synthesized batch ofthe peptide product.

In the method of the present invention, the peptide product comprises anunknown amount of at least one impurity, which cannot be quantitativelyseparated from the peptide product by a chromatographic procedure orwhich co-elutes with other impurities or ingredients present in thepeptide product sample. In some cases, the peptide product compositioncomprises unknown amounts of one impurity. In other cases, the peptideproduct composition comprises unknown amounts of at least two, e.g. 2,3, 4, 5 or even more impurities which cannot be quantitatively separatedfrom the peptide product by a chromatographic procedure or whichco-elute with other other impurities or ingredients present in thepeptide product sample. Preferably, the at least one impurity cannot bequantitatively separated from the peptide product.

The term “chromatographic procedure” involves a chromatographicprocedure suitably for the purification of peptide products, includinge.g. ion exchange chromatography, hydrophobic interactionchromatography, affinity chromatography, size exclusion chromatography,and particularly high performance liquid chromatography (HPLC) and moreparticularly Reverse Phase HPLC, or combinations of several procedures.

The impurity which is determined by the method of the present inventioncannot be quantitatively separated from the peptide product or fromother ingredients of the composition. This means, the impurity co-elutesor substantially co-elutes with the desired peptide product or withother impurities or ingredients after a chromatographic separationprocedure, particularly after a chromatographic separation procedure asdescribed above in such a way that quantification by optical methods,e.g UV- or fluorescence detection is not possible. This means, that theimpurity cannot be separated from the desired peptide product or fromother ingredients including impurities by reasonable efforts and itsquantitative amount in a given batch of the peptide product compositionhas to be quantitatively determined by the method of the presentinvention.

Step (a) of the inventive method comprises providing a peptide productcomposition comprising an unknown amount of at least one impuritywherein that impurity cannot be separated from the peptide product oranother ingredient of the composition by a chromatographic procedure asdescribed above. The peptide product may e.g. be a product for use inpharmaceutical applications, e.g. for use as a medicament in human orveterinary medicine.

Step (b) of the inventive method involves providing at least one sampleof the peptide product composition without added impurity and optionallyat least one further sample of the peptide product composition with aknown amount of added impurity. In a preferred embodiment, step (b)involves providing at least three samples of the peptide productcomposition wherein the first sample comprises the peptide productcomposition without added impurity in wherein at least two furthersamples comprise the peptide product composition and each of thesesamples additionally comprises a different known amount of addedimpurity.

At least one sample is a sample which comprises the peptide productcomposition without added impurity, i.e. an un-spiked sample comprisingan unknown amount of the impurity to be determined. In addition to thissample, at least one, e.g. at least two, three or four further samplesmay be provided which are spiked samples comprising the peptide productcomposition with the unknown amount of impurity and additionally withdifferent known amounts of added impurity to be determined. These spikedsamples may be prepared by adding the impurity to the un-spiked peptideproduct composition samples from a stock preparation of the impurity,preferably from at least two stock preparations comprising differentconcentrations of the impurity. The stock preparations may be present inany suitable form, e.g. preparations in dry or liquid form. Preferablythe stock preparations are stock solutions.

The tested samples comprise the peptide product with an unknown amountof the impurity to be determined and optionally the added impurity.These compounds are present in concentrations, which allow analysis bymass spectrometry. The concentration of the peptide product may vary toa great extent. It may be e.g. in the range of 0.001-50 mg/ml,preferably 0.01-10 mg/ml and more, preferably 0.02-5.0 mg/ml, forexample about 1.0 mg/ml. In the spiked samples, the amount of addedimpurity is preferably in the range of 0.1%-5%, preferably from 0.1-2%based on the concentration of the peptide product in the sample.

In a preferred embodiment, the method of the invention comprises thetesting of an un-spiked peptide product sample together with at leastone, preferably at least two, three or four or even more peptide productsamples spiked with at least one impurity. In a different embodiment,the method of the invention involves the testing of only an un-spikedpeptide product sample. The unknown amount of impurity present in theun-spiked sample may be determined by reference measurements of spikedsamples and/or by comparing the measured value of the impurity in theun-spiked sample with a reference, e.g. a standard or calibration curve.

Step (c) of the inventive method comprises quantitatively determiningsaid impurity in said at least one sample from step (b) by highresolution mass spectrometry. In addition to mass spectrometry, thedetermination may involve a prior chromatographic procedure, e.g. inorder to separate other impurities from the peptide product or fromother ingredients of the composition. Preferably, mass spectrometry iscombined with HPLC. Each sample may subjected to several individualdeterminations in order to increase the accuracy of the measurement.

Mass spectrometry is based on a measurement of the mass-to-charge ratioof charged particles. In a typical mass spectrometry procedure, thesample is loaded onto the mass spectrometry instrument and volatilized.

The sample components are ionized and the resulting ions are separatedin the mass analyzer by electromagnetic fields. The resulting ions aredetected and the signal is processed into a mass spectrum. For theionization of peptide products, electrospray ionization (ESI) andmatrix-assisted laser desorption/ionization (MALDI) may be used. Theresulting ions may be detected by highly sensitive methods such asOrbitrap or Fourier Transform (FT)-Ion Cyclotron Resonance (ICR)detection systems.

According to step (c), at least one peak associated with the impurity tobe determined is analyzed by mass spectrometry. This peak may be derivedeither from a deconvoluted or non-deconvoluted mass spectrum.Preferably, the method involves the determination of one or severalmonoisotopic peaks associated with the impurity to be determined. In thecase of the impurity Di-Ser(33)-AVE0010, a quadruply charged peak of anon-deconvoluted mass spectrum at e.g. 1237.1529 Da or of thedeconvoluted mass spectrum at 4944.5822 Da may be used. In case of theimpurity Di-Ala(35)-AVE0010, a quadruply charged peak of thenon-deconvoluted mass spectrum at 1233.1550 Da or of the deconvolutedmass spectrum at 4928.5908 Da may be analyzed.

The at least one peak to be analyzed may be identified in a massspectrum of the impurity using a high resolution setting and a highlysensitive detection as described above.

Step (d) comprises calculating the amount of the impurity in the peptideproduct composition based on the mass spectrometry analysis results. Ina preferred embodiment, the calculation is carried out by a methodcomprising a regression analysis, particularly a linear regressionanalysis. The regression analysis may comprise generating a curve, e.g.a straight line, from the measured values of the impurity in at leasttwo spiked samples, i.e. samples comprising the peptide product with theunknown amount of impurity and having added the two different knownamounts of impurities (or from reference values of such samples whichhave been generated in standard or calibration measurements). Into thiscurve, e.g. a straight line, the measured value of the impurity in theun-spiked sample is inserted thereby allowing quantitative determinationof the unknown amount of the impurity present in that sample.

Preferably, the calculation is carried out according to the linearstraight line equation:

y=ax+b

wherein y corresponds to the determined peak area of the impurity in asample (either spiked or un-spiked sample), x corresponds to the knownadded amount of the impurity in a spiked sample, a is the straight lineslope and b is the intercept with the y-axis, which corresponds to themeasurement signal of the impurity in a sample without added impurity(x=0). The quantitative amount of impurity in the un-spiked sample x_(t)may be obtained using the obtained regression parameters as follows:

x _(t) =b·a ⁻¹

The invention also encompasses a reagent kit for determining the amountof impurities in a Lixisenatide product which comprises at least onestock preparation of Di-Ser(33)-AVE0010 and/or Di-Ala(35)-AVE0010 andoptionally further reagents such as solvents, buffers etc. The reagentkit may be used in a method for the quality control of a Lixisenatideproduct for the quantitative determination of the amount ofDi-Ser(33)-AVE0010 and/or Di-Ala(35)-AVE0010 in said Lixisenatideproduct. Preferably, the quantitative determination is carried accordingto a mass spectrometric method, e.g. a mass spectrometric method asdescribed above.

The present invention is explained in more detail by the followingFigures and Examples.

LEGENDS OF FIGURES

FIG. 1 shows the co-elution of the peaks of Lixisenatide (AVE0010),Di-Ser(33)-AVE0010 and Di-Ala(35)-AVE0010 in an HPLC chromatogram.

FIG. 2 shows the mass spectrum of the quadruply charged molecular ionsof AVE0010 and the impurities Di-Ser(33)-AVE0010 and Di-Ala(35)-AVE0010.

FIG. 3 shows a comparison of mass spectra of un-spiked and spiked (1% ofeach impurity) AVE0010 preparations.

FIG. 4 shows an expanded view of the mass range of Di-Ser(33)-AVE0010 atthe resolution setting of 60,000 and 100,000 respectively at m/z400 andthe theoretical calculated pattern of overlapping species.

FIG. 5 shows deconvoluted mass spectra with theoretical calculatedpatterns of overlapping species.

FIG. 6 shows an expanded view on the impurity peak used for thequantification of Di-Ser(33)-AVE0010.

FIGS. 7A and 7B show an extracted single ion chromatogram of a quadruplycharged isotope of Di-Ala(35)-AVE0010 (FIG. 7A) and Di-Ser(33)-AV0010(FIG. 7B) in a test sample as a non-deconvoluted mass spectrum.

FIGS. 8A and 8B show an extracted single ion chromatogram ofDi-Ala(35)-AVE0010 (FIG. 8A) and Di-Ser(33)-AV0010 (FIG. 8B) in a testsample as a deconvoluted mass spectrum.

FIG. 9 shows an example for calculation of the concentration of theimpurity Di-Ala(35)-AVE0010 in a test sample.

EXAMPLES

Analytical Procedure for Quantification of Di-Ser(33)-Lixisenatide andDi-Ala(35)-Lixisenatide by HPLC/MS

1.Materials and Methods

1.1 Reference Materials

Lixisenatide (AVE0010) Reference material; produced by solid phasesynthesis, followed by purification.

Di-Ser(33)-AVE0010 Reference material; produced by solid phase synthesisprocess with double coupling steps of the protected amino acid Serine atposition 33, followed by purification.

Di-Ala(35)-AVE0010 Reference material; produced by solid phase synthesisprocess with double coupling steps of the protected amino acid Alanineat position 35, followed by purification.

An un-spiked sample preparation of Lixisenatide (containing unknownamounts of impurities DiSer(33)-AVE0010 and DiAla(35)-AVE0010) wasprepared at a concentration of 1.0 mg Lixisenatide/ml.

Four spiked sample preparations for each of the impurities were preparedby incorporating 0.2%, 0.4%, 0.6% and 0.8% by weight of the impuritiesDi-Ser(33)-AVE0010 or Di-Ala(35)-AVE0010, respectively, in a samplepreparation with 1.0 mg Lixisenatide/ml.

1.2 Analytical Conditions

A gradient high performance liquid chromatographic system consisting ofa binary HPLC pump, an autosampler, column oven, and a high resolutionmass spectrometer e.g. LTQ-Orbitrap (ThermoFisher) with a resolutionsetting of 100.000 (at m/z 400), or equivalent was used.

Chromatography was performed with a C18 Reversed Phase analytical HPLCcolumn (Jupiter C18 3 μm 300 A) with a length of 150 mm and an internaldiameter of 2.0 mm.

Mobile Phase A consisted of 850 volume parts water, 150 volume partsacetonitrile and 1 volume part trifluoroacetic acid. Mobile Phase Bconsisted of 250 volume parts water, 750 volume parts acetonitrile andone volume part trifluoroacetic acid. The gradient was set as follows:

Time Mobile phase A Mobile Phase B [minutes] [%] [%] 0.00 77.5 22.517.50 28.0 72.0 17.51 77.5 22.5 26.00 77.5 22.5

The mass spectrometer was operated in positive electrospray ionizationmode and scanned in a way to measure the ion signals from the analytesto determined.

The test conditions were as follows:

Flow rate: 0.25 mL/min Injection volume: 10 μL Autosampler Setautosampler temperature at +10° C. ± 2° C. temperature: Column Set oventemperature at +25° C. ± 2° C. temperature: Ionization method: ESIpositive Measurement/ ITMS: mode: positive Detection: mass range: 700.0-2000.0 FTMS: mode: positive mass range(SIM) 1232.0-1239.0resolution: 100,000 Divert waste    0-1.5 min Valve: inject 1.5 2.25.5min PDA total scan (UV), optional

The procedure was based on the methods described in Ph. Eur 7.0 (2011)(2.2.43 Mass Spectrometry) and USP 34 (2011) (<736> Mass Spectrometry).

1.3 Integration

Determinations were performed either from the deconvoluted mass spectraor the non-deconvoluted mass spectra of the quadruply charged ions[M+4H⁺]⁴⁺.

After selection of the accurate mass of a single isotopic peak of thepattern:

-   -   a) using a quadruply charged peak of the non deconvoluted mass        spectra:

DiSer(33)-AVE0010: e.g. 1237.1529 Da DiAla(35)-AVE0010: e.a. 1233.1550Da,

-   -   or b) using the deconvoluted mass spectra:

DiSer(33)-AVE0010: e.g. 4944.5822 Da DiAla(35)-AVE0010: e.g. 4928.5908Da,

extracted ion chromatograms were performed. Only the isotope of interestwas extracted. The peaks from the extracted ion chromatograms wereintegrated.

2. Results

FIG. 1 shows the co-elution of the peaks for Lixisenatide and theimpurities Di-Ala(35)-AVE0010 and Di-Ser(33)-AVE0010 in an HPLCchromatogram. Due to these overlaps in the chromatographic peaks,Di-Ala(35)-AVE0010 and Di-Ser(33)-AVE0010 constitute impurities whichcannot be quantitatively separated from the desired peptide productAVE0010 by a chromatographic procedure.

For the method development some specific features of mass spectrometryhad to be taken into account. The mass spectrum of Lixisenatide showsbesides the multiply protonated species typically clustering withcations that are ubiquitous in solution, e.g. sodium or potassium. FIG.2 shows the spectrum section of the quadruply charged ions of AVE0010which overlap with the mass spectra of the impurities and therebyobscure the analyte.

In FIG. 3 an AVE0010 sample spiked with 1% of the two impurities iscompared with an un-spiked sample containing yet a low amount of theimpurities. In the expanded view of the inset can be seen, that theremay be an overlap of the molecular patterns with underlying clusters. Inthe case of Di-Ser(33)-AVE0010 the mass increase relative toLixisenatide is 87 Da. On the other hand, Lixisenatide can cluster with4 sodium ions leading to a mass shift of 88 Da. These two patternsoverlap and have to be separated mass-spectrometrically otherwise theDi-Ser-AV0010 impurity cannot be quantified specifically.

As can be seen in FIG. 4, the overlapping peaks were not separated witha resolution setting of 60.000 but only with a resolution setting of100.000. In the lower part of FIG. 4 the theoretical spectrum ofDi-Ser-AVE0010 overlapping with the sodium cluster of Lixisenatide isshown. Such high resolutions are preferably delivered by Fouriertransform mass spectrometry, either FT-ICR or as used here by anFT-Orbitrap mass spectrometer.

After deconvolution and centroiding of the spectra, these two speciesmay be separated mass-spectroscopically, c.f. FIG. 5.

In FIG. 6 all measured individual mass spectra of the completechromatographic peak have been added. The mass peak used for themeasurement of Di-Ser(33)-AVE0010 is shown in an expanded view. It issufficiently distant from the peak corresponding to the sodium clusterof AVE0010 to allow an accurate measurement. All analyte mass peaks fallwithin a narrow mass window, showing the mass stability of theinstrument, well separated from any interfering peak. Therefore thismethod is suited to determine specifically the amounts ofDi-Ser(33)-AVE0010 and Di-Ala(35)-AVE0010 in a Lixisenatide peptideproduct.

The extracted ion chromatograms of a quadruply charged isotope of DiAla(35)-AVE0010 and Di-Ser(33)-AV0010 in a test sample are shown asnon-deconvoluted mass spectra in FIGS. 7A and 7B and as deconvolutedmass spectra in FIGS. 8A and 8B.

The amount of Di-Ser(33)-AVE0010 and Di-Ala(35)-AVE0010 may becalculated by a linear regression analysis. By using each injection ofthe spiked and un-spiked test solutions, a regression curve may beestablished according to the equation:

y=ax+b

-   -   a=Slope    -   y=Peak area of test solutions (un-spiked and spiked test        solutions)    -   x=Added amount of the Di-Ser(33)-AVE0010 or Di-Ala(35)-AVE0010        in test solutions (unspiked and spiked test solutions).    -   b=Intercept

The concentration x_(t) of Di-Ser(33)-AVE0010 and Di-Ala(35)-AVE0010 inthe test sample may be calculated using the regression parametersobtained as follows:

x _(t) =b·a ⁻¹

FIG. 9 shows an example for calculation of the concentrationDi-Ala(35)-AVE0010 in the test sample.

The final result was determined as the arithmetic mean of all individualdeterminations expressed in percent of Lixisenatide. In the specificcase, the mean value of the impurity Di-Ala(35)-AVE0010 in the samplewas determined as 0.4260% based on the amount of Lixisenatide.

1. A method for the quantitative determination of an impurity present ina peptide product composition, comprising the steps: (a) providing apeptide product composition comprising a peptide product and an unknownamount of at least one impurity, wherein said impurity cannot beseparated from the peptide product or another ingredient of thecomposition by a chromatographic procedure, (b) providing at least onesample of the peptide product composition without said impurity addedand optionally at least one further sample of the peptide productcomposition with a known amount of said impurity added, (c)quantitatively determining said impurity in said sample from step (b) bymass spectrometry, and (d) calculating the amount of said impurity inthe peptide product composition based on the results of (c).
 2. Themethod according to claim 1 wherein step (b) comprises providing atleast three samples of the peptide product composition, wherein a firstsample comprises the peptide product composition without said impurityadded, and wherein at least two further samples comprise the peptideproduct composition each with a different known amount of said impurityadded.
 3. The method of claim 1, wherein the peptide product has alength of from 5-100 amino acids.
 4. The method of claim 1, wherein thepeptide product has been chemically synthesized, particularly by a solidphase synthesis procedure, or produced by recombinant DNA processes. 5.The method of claim 1, wherein the peptide product composition is apharmaceutical formulation or a composition intended for the manufactureof a pharmaceutical formulation.
 6. The method of claim 1, wherein thepeptide product is an exendin peptide, particularly Lixisenatide(AVE0010).
 7. The method of claim 1, wherein the impurity is a peptideimpurity.
 8. The method of claim 1, wherein the impurity cannot bequantitatively separated from the peptide product or from anotheringredient of the composition by an HPLC procedure, particularly by aReverse Phase HPLC procedure.
 9. The method of claim 1, wherein thepeptide product comprises unknown amounts of at least 2 impurities whichcannot be quantitatively separated from the peptide product or fromanother ingredient of the composition by a chromatographic procedure.10. The method of claim 1, wherein the impurity is added to at least onepeptide product composition sample from a stock preparation, preferablyfrom at least two stock preparations comprising different concentrationsof the impurity.
 11. The method of claim 1, wherein at least 3 or 4further samples with different known amounts added impurity are providedand subjected to mass spectrometry determination.
 12. The method ofclaim 1, wherein the mass spectrometry is high resolution massspectrometry.
 13. The method of claim 1, wherein the calculationcomprises a linear regression analysis.
 14. The method of claim 13,wherein the calculation is carried out according to the equation:y=ax+b wherein a=slope y=determined peak area of the impurity in asample x=added amount of the impurity in a sample b=intercept and theunknown amount of the impurity x_(t) is obtained as follows:x _(t) =b·a ⁻¹
 15. The method of claim 12, wherein said high resolutionmass spectrometry comprises Fourier Transform mass spectrometry.